1) Comparison of first- and second-generation RT-QuIC assays. Previous studies have shown that RT-QuIC testing of human cerebrospinal fluid (CSF) is highly sensitive and specific in discriminating sporadic CJD patients from those without prion disease. Using CSF samples from 113 CJD and 64 non-prion disease patients, we provided the first direct and concurrent comparison of our improved RT-QuIC assay to our previous assay that is similar to those commonly used internationally for CJD diagnosis. This extended comparison demonstrated a 21% increase in diagnostic sensitivity, a 2-day reduction in average detection time, and 100% specificity. 2) Diagnostic value of RT-QuIC analyses of CSF and nasal swabbings. In collaboration with the University of Verona and the Istituto Superiore di Sanit, Rome, we have also shown previously that RT-QuIC also detects minute amounts of the disease-specific pathological prion protein(PrPCJD)in olfactory mucosa (OM) samples. We have now assessed RT-QuIC accuracy in the antemortem diagnosis of CJD by using the RT-QuIC assay on CSF, olfactory mucosa samples, or both. We collected CSF and OM samples from 86 patients with a clinical diagnosis of probable (n=51), possible (n=24) or suspect (n=11) CJD and negative controls (50 OM and 54 CSF). CSF and OM samples were analyzed by conventional RT-QuIC. CSF samples were further tested using improved RT-QuIC conditions. We also evaluated the diagnostic performance of a novel, easy to use and gentle flocked swab for sampling of olfactory mucosa (OM). All 61 confirmed sporadic CJD patients were RT-QuIC-positive using OM, CSF, or both, giving an overall RT-QuIC diagnostic sensitivity of 100% (CI, 93-100%). All non-CJD patients (n=71 for CSF and n=50 for OM) were negative giving 100% specificity (CI, 914 or 91-100%, respectively). Of 8 symptomatic patients with various mutations causing either CJD or GSS, 6 were positive and 2 were negative, giving a sensitivity of 75% (, CI, 36-96%). We proposed a diagnostic algorithm for sCJD that combines CSF and OM RT-QuIC testing to provide virtually 100% diagnostic sensitivity and specificity in the clinical phase of the disease. 3) Further clinical validation of improved, second generation RT-QuIC. In further collaborative studies we helped major human prion disease diagnostic centers perform extensive analyses of the diagnostic performance of our second-generation RT-QuIC assay. These centers included the US National Prion Disease Pathology Surveillance Center, the UK National CJD Research and Surveillance Unit, the Italian IRCCS Institute of Neurological Sciences, Bologna and Foundation Carlo Besta Neurological Institute, Milan. We provided training and key reagents for these studies. The findings provided strong confirmation of the improved, state-of-the-art diagnostic accuracy of our assay and extended the applications to the detection and discrimination of various human prion diseases and variants thereof. 4) An RT-QuIC assay for tauopathy. The diagnosis and treatment of diseases involving tau-based pathology such as Alzheimer disease and certain frontotemporal dementias is hampered by the inability to detect pathological forms of tau with sufficient sensitivity, specificity and practicality. In these neurodegenerative diseases tau accumulates in self-seeding filaments. For example, Pick disease (PiD) is associated with frontotemporal degeneration and accumulation of 3-repeat (3R) tau isoforms in filaments constituting Pick bodies. Exploiting the self-seeding activity of tau deposits, and using a 3R tau fragment as a substrate, we developed an assay (tau RT-QuIC) that can detect tau seeds in 2 l aliquots of PiD brain dilutions down to 10-7-10-9. PiD seeding activities were 100-fold higher in frontal and temporal lobes compared to cerebellar cortex. Strikingly, this test was 103- to 105-fold less responsive when seeded with brain containing predominant 4-repeat (4R) tau aggregates from cases of corticobasal degeneration, argyrophilic grain disease, and progressive supranuclear palsy. Alzheimer disease brain, with 3R+4R tau deposits, also gave much weaker responses than PiD brain. When applied to cerebrospinal fluid samples (5 l), tau RT-QuIC analyses discriminated PiD from non-PiD cases. These findings demonstrate that abnormal tau aggregates can be detected with high sensitivity and disease-specificity in crude tissue and fluid samples. Accordingly, this tau RT-QuIC assay exemplifies a new approach to diagnosing tauopathies and monitoring therapeutic trials using aggregated tau itself as a biomarker.